The fatty acid composition of total lipids and the adrenoceptor mediated activation of glycogenolysis were studied in isolated hepatocytes from rats maintained on a control diet or on an essential fatty acid (EFA)-free diet. In cells from rats on the EFA-free diet there was a marked reduction in linoleic and arachidonic acid (AA) contents and an increase in eicosatrienoic, oleic and palmitoleic acid contents compared to controls. In freshly isolated cells from both groups, the adrenergic activation of glycogen phosphorylase was mediated only by alpha1 and not by beta-receptors. When control cells were preincubated in a serum-free buffer for 4 hours before testing, the alphaadrenergic component was blunted and a cAMP dependent beta-adrenergic component of the phosphorylase response emerged. A similar 4 hr incubation of EFA-deficient cells resulted in a reduced alpha but continued absence of a beta-receptor response. A beta-receptor response of these 4 hr cells could be restored by in vivo replacement of the EFA-deficient diet with control diet for the last 4 weeks prior to the experiment, but not by the in vitro exposure of EFA-deficient cells to 10 uM AA throughout the 4 hr incubation period. Extending previous observations, the present results suggest that the time- dependent emergence of beta-adrenergic glycogenolysis, but not the parallel reduction of the alpha-adrenergic response, is mediated by AA or its metabolite(s), which probably act by facilitating the G-protein dependent coupling of beta-receptors. Inverse regulation of hepatic alpha1 and beta2 receptors has been demonstrated in rat liver cells in primary culture DNA solution hybridization assays for alpha1 and beta2 receptors have been developed to analyze the transcriptional regulation of the two receptors in the above experimental model.